Fermentative carbomycin production



Sept. 1, 1959 J. F. PAGANo ETAL EERMENTATIVE cARBoMYcIN PRODUCTIONOriginal Filed July 17. 1952 me/whole www O\KR NCF-R AWHE U- @OCS 202.99www Se 8: 8.. 3.88 SSSSn ANTU POUE. .DDH IO- 4 .F D. f Hw J4 H 56M olAE8u JRJL a -ov m m. 18m, N m w IDF 18 I8 18.

United States Patent FERMENTATIV E CARBOMYCIN PRODUCTION Joseph F.Pagano, Bound Brook, Richard Donovick, Westfield, .lames D. Dutcher, NewBrunswick, and Leon J. Heuser, Princeton, NJ., assignors to OlinMatheson Chemical Corporation, New York, N.Y., a *corporation ofVirginia Continuation of application Serial No. 299,368, July 17, 1952.This application June 4, 1956, Serial i No. '589,324

6 Claims. t (Cl. H5-80) This application is a continuation of ourapplication Serial No. 299,368, filed July 17, 1952, now abandoned.

This invention relates to a new antibiotic and to its production. Moreparticularly, it relates to a new antibiotic in various forms, and toprocesses for producing it by fermentation, as well as concentratingand/ or purifying it, isolating it, and producing salts thereof. Theinvention embraces the antibiotic and its salts in solution form, asconcentrates, and in pure crystalline form.

The antibiotic of this invention is formed by the cultivation, underco-ntrolled co-nditions, of a hitherto undescribed species ofStreptomyces isolated from a soil sample obtained in North Charleston,North Carolina, and referred to hereinafter as Streptomycesthermotolerans. A culture o-f the living organism has been deposited andmade part of the Stock Culture Collection of the New Jersey AgriculturalExperiment Station, New Brunswick, New Jersey, where it has beenassigned the number, 3628. A culture of the living organism has alsobeen deposited and made part of the American Type Culture Collection,Washington, D C., where it has been designated as ATCC 11,416. Culturesare available from either source.

For isolating and characterizing the microorganism, a portion of thesoil sample (1 g. approx.) is diluted in sterile water, plated on anagar containing in distilled water: sucrose, 1%; citric acid, .12%;(NHQZHPOQ 04%; KCl, 008%; MgCl2-6H2O, .0418%; MnCl2.6H2O, .0036%;FeCl3.6H2O, .0023%; ZnCl2, 002.1%; CoClzHzO, .0004%; galactose, 1.0%;and agar, 1.5%; which is then incubated at 26 C. for 10 days. A colonyof the newly-isolated Streptomycete, selected from the organisms on theplates, when streak-tested against bacteria, has been found to showactivity against Micrococcus pyogenes var. aureus, Aerobacilluspolymyxa, and Streptococcus faecalis.

In the following description of colonies of the organism after 6 daysincubation at 26 C. in various agar media, the designated colors arebased on Ridgway, Color Standards and Color Nomenclature, Washington,D.C., 1912:

(In yeast-beef agar, consisting of: yeast extract, 0.3%; beef extract0.15%; peptone, 0.6%; dextrose, 0.1%; agar, 1.5% and distilled water.)Growth is good. Aerial mycelium and spores are cartridge buff to palesmoke grey. The colonies are convex and opaque with edge lacerate.Sporulation is heavy; aerial mycelium slight with short irregularprotuberances. Reverse color is tawny buff. Agar diffusible pigmentproduced which is pinkish buff. The vegetative mycelium is 0.5-0.7microns in diameter. The spores are 0.5 x 1 0-1.5 microns in diameter.

(In Czapek-Dox agar, consisting of NaNO3, 0.3%; KH2PO4, 0.1%; KCl,0.05%; MgSO4.7H2O, 0.05%; FeSO4.7I-12O, 0.001%; glucose, 4.0%; agar,1.5%; and distilled water.) Colonies are circular, finely granular, lowconvex with lacerate edge. Spores are pale pinkish butt" and reversecolony is primuline yellow No exopigment is produced.

2,902,412 Patented Sept. 1, 1959 (In Sabourauds agar, consisting of:neopeptone, 1.0%; dextrose, 4.0%; agar, 1.5 and distilled water.)Colonies are circular, finely granular, low convex with lacerate edge.Spores are Roods brown to pale pinkish buff and reverse colony is warmsepia. Agar diffusible pigment produced which is clay color.

(In soybean infusion agar, consisting of: soy infusion (boiled 30minutes, filtered) 2%; dextrose, 0.2%; sodium chloride, 0.5%; agar,2.0%; and distilled water (adjusted to pl-I 7 before sterilization).)Colonies are circular, finely granular, opaque, convex with lacerateedge, spores are white to pale smoke grey. The reverse colony isbuckthorn brown in the center with outer edges warm buff. No exopigmentis produced.

(In Henrici agar, consisting of: casein hydrolysate, 0.5%; glycerine,0.5%; K2HPO4, 0.2%;. MgSO..7H2O, 0.2%; FeSo4.7H2O, 0.2%; agar, 1.5%; anddistilled water.) Colonies are white with a raised crown which is pallidmouse grey. The reverse colony is clove brown with a circular edge whichis blackish brown No. 3. Exopigment is buffy brown.

The above description of the organism used to obtain the antibiotic ofthis invention is merely illustrative, and itis to be understood thatthis invention is not limited to the use of that organism or toorganisms fully answering that description, but includes, inter alia,mutants produced from the described organism by mutating agents, such asX-radiation, ultra-violet radiation and nitrogen mustards.

The microorganism is capable of assimilating the following carbonsources in a basal medium containing (NHQZSO, as a source of nitrogen;rhamnose, xylose, glucose, galactose, fructose, mannose, lactose,maltose, sucrose, rainose, glycerol, mannitol, dextrin, inulin, starch,inositol and ammonium tartrate. The following carbon compounds supportgrowth poorly; arabinose, sorbitol, sodium citrate. Growth is notsupported by any of the following carbon compounds: dulcitol, salicin,sodium acetate, ammonium formate, ammonium oxalate.

In a basal medium containing starch as a source of carbon, the followingnitrogen sources will support growth: ammonium sulfate, sodium nitrite,and asparagine; tyrosine supports growth poorly: and acetamide will notsupport growth.

The microorganism is also identified by its ability to grow at 26 C. and37 C. on yeast-beef, soybean meal, and Sabourauds agar. It does notproduce indole and does not reduce nitrate, but will liquefy gelatinwith formation of brown pigment, and cause litmus milk to become acid.

The antibiotic of this invention has been found to possess a wideantibiotic spectrum, in vitro tests against a variety of organisms andin general to be effective in inhibiting the growth of gram positivebacteria and mycobacteria. Thus, among the organisms failing toprop-agate in the presence of the antibiotic are M z'crococcus pyogenesvar. aureus, Streptococcus pyogenes, Streptococcus faecalis, Diplococcuspneumoniae type 2, Diplococcus pneumoniae type 3, Bacillus subtilis,Clostridium septicum, Klebsiella pneumoniae, Escherichia coli,Aerobactcr uerogenes, Salmonella typhosa, Salmonella schottmulleri,Shigella dysenteriae, Shigella sonnet', Proteus vulgaris, Pseudomonasaeruginosa, and Mycobacterium tuberculosis var. bovz's BCG. When in purecrystalline form, the antibiotic of this invention possesses a highorder of activity, comparable to that of chloramphenicol when testedagainst meningopneurnonitis virus and about 4 times that ofchloramphenicol when tested against Rickertsz'a rckettsi. Furthermore,due to its solubility in oil, the antibiotic of this invention isespecially adapted for use in various delayed-action oil-baseformulations.

'I'he data following shows the potency of antibiotic-containing brothsobtained in the practicel of this invention,

measured -in dilution units (which, as used herein, is the reciprocal ofthe highest dilution of the broth which completely inhibits the growthof a test organism, that organism being Microcoecus pyogenes var. aureus209P unless otherwise specified). In these tests, fermentation wasallowed to proceed for the specified periods in a soybean-meal nutrientmedium (adjusted to pH 7 after sterilization) consisting of: soybeanmeal, 3%; dextrose, 2%; CoCl2.6H2O, 0.0005 and CaCO3, 0.1%, in distilledwater: and the dilution units were determined against the selected testorganisms.

DILUTION UNITS pH of fermentation at 4 days: 7.9. pH of fermentation at7 days: 7.7.

'Ihe antibiotic of this invention is preferably produced by submergedaerated culture of the organism to yield broths (in large scalefermentation) having potencies up to about 2000 or more d.u./m., but mayalso be produced by surface culture, with aeration provided by merelyexposing the surface to a sterile air supply. In either case, sources ofcarbon for energy and nitrogen for growth are included in the nutrientmedium.

As the energy-source material one may use: a carbohydrate, such asstarch, soluble starch, dextrose, sucrose, and maltose: a sugar alcohol(eg. glycerol); or a lipid, such as (1) fat acid, (2) a fat or (3) amixture of such materials. Illustrative fats are lard oil, soybean oil,

linseed oil, cottonseed oil, peanut oil, coconut oil, corn oil, castoroil, sesame oil, crude palm oil, fancy mutton tallow, sperm oil, oliveoil, tristearin, tripalmitin, triolein and trilaurin; and illustrativefat acids are acetic, propionic, butyric, stearic, palmitic, oleic,lauric, linoleic, and myristic acids. Preferred as the energy-sourcematerials are the carbohydrates, especially dextrose.

The sources of nitrogenous, growth-promoting factors are those normallyemployed in such processes. They may be natural organics (e.g. soybeanmeal, corn steep liquor, meat extract, casein, fish meal, liver cake,and/or distillers solubles) or synthetics such as inorganic nitrates orammonium compounds. Corn steep liquor because of the wide variety ofsubstances contained therein, is a valuable addition to the fermentationmedium.

The media used in the process of the invention may contain precursors inaddition to the nutrient components, to obtain other valuable products.-For example, an assimilable source of cobalt may be included wherecobalamines (vitamin B12 and vitamin B12-like products) are desired, andthese by-products then recovered by conventional methods. Or steroidprecursors, such as progesterone or Reichsteins Compound S or S acetate,may be added to obtain a steroid oxidized in the 11 position.

As in most fermentation processes, the process of the present inventionis desirably carried out using a liquid medium containing mineralcomponents enhancing growth of the organism, for example, sources ofpotassium calcium, magnesium, sulfur, iron, other trace elements, andphosphate. These components are desirably added to the medium unlessalready present therein as a component (e.g. impurity) of the crudecartonor nitrogensource material (e.g. corn steep liquor).

.In large scale fermentation by submerged aerated culture, the pH of themedium is preferably adjusted if necessary to about 7 (although a pHabout 5 to 9 may be used) by addition of buffering agents, the pHtending to become slightly alkaline (pH about 7.5-8.5) as fermentationproceeds. Fermentation temperatures from about 20 C. to about 40 C. maybe used, with a temperature of about 25 C. preferred. The stirring maybe effected by mechanical agitation at or more r.p.m. with aeration at asuperficial velocity up to about 3 or more meters per minute.

Small scale fermentation, for laboratory investigatio or for theproduction of inoculae for larger fermentations, may be conducted inshaker asks plugged with cotton. Thus, 250 ml. of an aqueous nutrientmedium containing: soybean meal, 3%; dextrose, 2%;

0.0005 and CaCO3, 0.1% is placed in a one-liter Erlenmeyer ask,sterilized in the usual manner (i.e. by autoclaving), then adjusted topH 7.0 with 12 N NaOH. The medium is then inoculated with the growth onan agar slant (either yeast-beef or soy infusion) of Streptomycesthermotolerans, and incubation is allowed to proceed for 48-96 hours ona reciprocating shaker oscillating at a rate of one-inch-strokes perminute. AOther media may be used, such as aqueous media containing: (A)beef extract, 0.15%; yeast extract, 0.15%; peptone, 0.5%; dextrose,0.5%; sodium chloride 0.35%; KLJ-1F04, 0.368%; KH2PO4, 0.132%; (B)cerelose, 1%; peptone, 1%; beef extract, 0.03%; yeast extract, 0.5%; and(C) dextrose, 4%; peptone, 1%.

The antibiotic of this invention may be obtained in crystalline formfrom the filtered fermentation broth by: (1) extracting with asubstantially water-immiscible organic solvent, such as benzene,chloroform, amyl acetate, ethyl acetate, butanol, amyl alcohol,trichlorethylene, and ether, at pH preferably about 6.6 or higher; (2)distilling off the organic solvent under vacuum in the presence ofwater, yielding a Water-insoluble oil fraction and a water-solublefraction; freeze drying the watersoluble fraction; and recrystallizingit from an organic solvent or solvent mixture, such as isopropanol, amixture of benzene and petroleum ether, aqueous methanol, ethanol oracetone. The product is colorless, and scparates in the form of prismsor needles; the crystals are not hydrated and after drying at 100 C. invacuo are not hygroscopic.

The crystalline antibiotic (free base) thus obtained is very soluble inmost organic solvents, inter alia, in ethyl acetate, glacial aceticacid, amyl acetate, butanol, ben- Following are the analytical dataobtained using the crystalline free base, dried in vacuo at 100 C.

Calculated for CHI-173151016: C, 59.48; -l-I, 8.68; N, 1.65;2,'(methoxyl), 7.31. Found: C, 59.24; H, 8.64; N, 1.76; methoxy, 7.33.

The crystalline material contains no detectable amount of halogen orsulfur. In dilute aqueous acid solution (0.05 N acetic acid), it (1)gives a precipitate with phosphotungstic acid, pioric acid, methylorange, and

Reinecke acid, (2) gives no precipitate with silver nitrate or mercuricchloride solution, (3) does not reduce Eehlings or Tollens reagent, (4)reduces potassium permanganate at room temperature, (5) consumes brominewater to form a turbid solution, (6) gives no color with ferrie chloridesolution, and (7) on addition of an equal volume of hydrochloricacid(conc), gives a purple color which fades to tan. i

The concentrate (i.e. the water-.soluble fraction'obtained byether-extraction of the broth ltrate followed by evaporation of theether in the `presence `of` water) may also be crystallizedV afterpurification byy adsorption. Thus the concentrate in acetone in benzeneis adsonbed. onto a silica gel column, and thecOIumnde- Veloped byelution with Aacetone-benzene solvent mirrtures containing a graduallyincreasing proportion of acetone. The active material, which is in asingle band, comes on? the'column with 30% acetone in benzene "(80% ofthe total activity adsorbed is thus recovered). The residue remainingafter allowing the solvent to evaporate from the eluate is readilycrystallized from isopropanol (Mr. zin-209 0.).

Acid-addition salts of the free base (mineral acid salts,

such-as' the hydrochloride, sulfate, phosphate; and organic acid saltssuch as the citrate, tartrate, gluconate, and p-tolueno sulfonate) maybe prepared using stand- 4ard procedures, water-soluble saltsofpharmacologicallyacceptable acids being preferred. Thus, thehydrochloride may be prepared by any of the following methods:

(a) Passing dry hydrogen chloride into an ether solution of the freebase.

(b) Dissolving the free base in absolute=methanol, adding one equivalentof methanolic hydrogen chloride, removing the lsolvent by evaporationunder avstrearn of .carbon dixoide, and washing the residue severaltimeswith dry ether.

(c) Dissolving the free base in absolute ether, adding a solution ofmethanolic hydrogen chloride dropwise until precipitation is complete,separating the precipitate by centrifugation and washing several timeswith dry ether.

The amorphous yhydrochloride (MP.` 1`57159 C. :in an open capillary inan oil bath) lis very soluble in methanol, ethanol, and acetone, solublein water, and insoluble Vin ethyl ether and hexane. lts specic rotationis [a]D23=-54i3 (absolute methanol, c=0.6).

The following `are additional characteristics of the crystalline freebase:

Craig distribution: Dissolved in an ethyl ether-2% aqueous NaH2P-O4mixture (pH 4.5) and run through a 24 tube Craig distribution system,bioassays and the ultraviolet absorption maximum at 241 mu show a peakat tube 6 with recoveries greater than 95%; and there is no indicationof the presence of another component.

Ultraviolet absorption: Measured .in 2% Nal-I2PO4, the ultravioletabsorption curve shows a maximum at 241 mp.,

and measured in methanol or ethanol, a maximum at 327.5 ma, and 240 mp.,

Infrared absorption: The infrared absorption spectrum has lbeen run asthe Nujol mull of the crystals; and Figure 1 is a graph of the data thusobtained, tabulated below.

. (Neuf-Substantially the same infrared absorption spectrlnn is obtainedusing a chloroform solution of the antibiotic.)

'1(0111F1) '1(0111-0 14cm-1) 2, 920 l, 160 928 1, 732 1, 129 916 1, 69()1, 110 j 873 1, 630 1, 095 860 1, 460 1, O77 837 1, 427 1,052 `821 1,375 l, 025 805 1, 300 l, O 781 1, 271 989 `74:2 1, 230 962 689 1, 193944 The behavior of the antibiotic of this invention in crystalline baseform when subjected to ascending paper w chromatography at 26 C. using aculture of Micrococcris pydgenes var. aureus as the test organism isgiven below. The position of the antibiotic (Rf) is located in each caseby a distinct zone of inhibition when placed on an agar medium seededwith the test organism.

DETERMINATION OF Rf Following are illustrative examples showing theprocess -by which the antibiotic of this invention may be produced.These examplesl Iare merely illustrative. and are Anot .to/,tbeconstrued as limiting the invention.

Example 1 PREPARATION OF INOCUDUM Stage I.-Yeast beef or soybeaninfusion agar slants of Streptomyces thermotolerans `are used toinoculate 500 ml. flasks, each containing ml. of the followinggermination medium: Soybean meal, 1.5%; glucose, 2%; sodium chloride,0.1%; calcium carbonate, 0.5%;

0.0005%; and tap 'water q.s. 100 m1. The medium is adjusted to pH6.8-7.2 with 12 N NaOH before sterilization in an autoclave lfor 30minutes at 121 C. The' asks are then inoculated and incubated at 25 C.for 72 hours on a reciprocating shaker having `a 2-inch throw and makingstrokes per minute.

Stage II.-Using the 72 hour-Stage I flasks, a 15 ml. sterile transfer ismade to 2 one-liter side arm shaker flasks, each containing 300 ml.ofthe sante germination medium as that used in Stage 1. The asks arethen incubated at 25 C. for 48 hours on a reciprocating shaker under thesame conditions as those described for Stage I.

FERMENTATION To l0 liters of a fermentation medium consisting of:Soybean meal, 3.0%; cerelose,.2.2%; sodium chloride, 0.1%; calciumcarbonate, 0.25%; cobalt chloride, 0.0005%; lard oil, 0.4; fand tapwaterqus., in an 18.9 liter stainless steel tank, lwhich has been steamsterilized for l5 minutes at 121 C. is added Ithe contents of-one StageII ask, obtained as described above. Fermentation -is then allowed toproceed at 25 C. with mechanical agitation at 300 r.p.rn., .and aerationat a rate of 28.3 liters/min. (superficial velocity-61 centimeters/min).A positive tank pressure is maintained at .35 kg./cm.2 gauge, with lardoil or prime burning oil being added reduction).

accadr ,as needed as an anti-foam agent. After 84 hours fermentation,the broth is adjusted to pH 2-3 with HZSO., (conc), then filtered usingHyilo (a filter aid). The filtered fbroth is extracted two times with 1Avolume ethyl ether at a pH 6.6. The ether is allowed to evaporate undervacuum in the presence of water to yield (1) a small amount of awater-insoluble oil and (2) a water-soluble fraction. When `thewater-insoluble oil is extracted with petroleum ether, a precipitatehaving a potency of 2500 d.u./mg., is formed. The water-solublefraction, on freeze drying and recrystallizat-ion from hot (60 C.)isopropanol yields a pure product having a potency of about 7-10,000d.u./mg.

ume of water at pH about 2.0-2.5 and the resulting aqueous solution isneutralized to yield the crystalline base, which is isolated byiiltration. On drying, 144g. base having a potency of 5000 d.u./mg. isobtained. Neutralization of thewater extract of the amyl acetate and thewater extractof the spent benzene yields approximate-I ly g. crystallineantibiotic. Recrystallization lof the antibiotic by dissolving thecrystals in hot 70% aqueous isopropanol, then adding water untilcloudiness occurs, yields 144 g. purified antibiotic having a potency of7000 d.u./mg. p

Following, in tabular form, is the fermentation data obtained inthecourse of the fermentations of Examples 1 and 2:

Example Z PREPARATION 0F INOCULUM Stage 1.-The procedure is the same asthat employed in Example 1, Stage I.

Stage 11.-A 72-hour-Stage I ask is used to inoculate an 18.9-literserrated bottle containing 12 liters of the same germination medium (themedium having been steam-sterilized at 121" C. before inoculation) asthat used in Stage I. The medium is adjusted to pH 6.8-7.2 with 12 NNaOH, then steam-sterilized for 11/2 hours at 121 C. Incubation is thenallowed to proceed at 25 C. Incubation is then allowed to proceed at C.for 48 hours, with aeration at a rate of one liter per liter of `mediumper minute.

FERMENTATION carbon steel tank, is added the contents of one Stage Il rbottle, obtained as described above. Fermentation is then allowed toproceed at 25 C. with mechanical agitation vat 120 r.p.m. and aerationat a rate of 368 liters/minute (superficial velocity-31 cms/sec) Apositive tank .pressure is maintained at .70 kga/cm.2 gauge, with lardoil or prime burning oil being added as needed as an antifoam agent.After 84 hours fermentation, the broth is adjusted to pH 2-3 with H2SO4(conc), then filtered using a filter aid (Hyo). The filtered broth isthen treated as in Example 1 to obtain the concentrate and thecrystalline antibiotic. (Alternatively, the crystalline antibiotic maybe obtained from the filtered broth as'follows: About 2630 liters offiltered broth are neutralized to pH about 7 and extracted with amylacetate in a two-stage counter-current system (4:1 volume reduction).The resulting amyl acetate solution (681 liters) is extracted in asimilar manner with waterv at pH 2.0-2.5 (4:1 volume The aqueoussolution (170 liters) is then extracted by agitating with about 35liters neutral benzene (pI-I about 7.0-7.5). After concentration toabout 4.5 liters, theV benzene extract is extracted with an equal vol-The antibiotic of this invention may be employed in the treatment ofvarious infections in man and animals, inter alia, infections by themicroorganisms listed hereinbefore in connection with its antibioticspectrum. The antibiotic may be employed per se as embodied in any ofthe usual formulations or dosage forms, for administration orally,parenterally, or topically. Also, the antibiotic may be administeredconcurrently with (or mixed with, in a suitable formulation) variousother antibiotics (c g. neomycin) or chemotherapeutic agents (eg.N-hydroxy- Z-pyridinethione) This invention may be variously otherwiseembodied within the scope of the appended claims.

We claim:

1. A process for producing an antibiotic, which comprises cultivatingStreptomyces thermotolerans under aerobic conditions in an aqueousnutrient medium containing assimilable sources of carbon and nitrogenuntil a substantial amount of antibiotic is produced, then recoveringthe antibiotic from the fermentation broth.

2. The process of claim 1 wherein submerged aerated conditions aremaintained.

3. The process of claim 2 wherein the temperature iS maintained at about20 to 40 C.

4. The process of claim 2 wherein the temperature is maintained at about25 C.

5. The process of claim 1, wherein thenutrient medium is maintained atpH about 5 to 9. y .L

6. The process of claim 1, wherein the nutrient medium is maintained atpH about 7.

1. A PROCESS FOR PRODUCING ANTIBIOTIC, WHICH COMPRISES CULTIVATINGSTREPTOMYCES THERMOTOLERANS UNDER AEROBIC CONDITIONS IN AN AQUEOUSNUTRIENT MEDIUM CONTAINING ASSIMILABLE SOURCES OF CARBON AND NITROGENUNTIL A SUBSTANTIAL AMOUNT OF ANTIBIOTIC IS PRODUCED, THEN RECOVERINGTHE ANTIBIOTIC FROM THE FERMENTATION BROTH.